BCL2-interacting Protein Research Articles

Endoplasmic reticulum stress and autophagy contributed to cadmium nephrotoxicity in HK-2 cells and Sprague-Dawley rats

Excessive accumulation of cadmium is known to cause nephrotoxicity by targeting renal proximal tubular epithelial cells. Studies showed an essential role of autophagy in cadmium-induced nephrotoxicity; however, its underlying mechanisms accompanied by autophagy are incompletely understood. Using an HK-2 human renal proximal tubular epithelial cell line as a study model, sustained exposure of cadmium chloride (CdCl2) was shown to cause cell viability loss, which was alleviated by inhibitors of autophagy but not apoptosis. Data from molecular and biochemical studies revealed an induction of autophagy proteins, intracellular acidic vesicles, and autophagic flux in CdCl2-treated cells. However, there was little sign of apoptosis-related changes. Pharmacological and genetic studies indicated an elevation of Endoplasmic Reticulum (ER) stress, Forkhead Box Class O (FoxO3a), Bcl-2 Interacting Protein 3 (Bnip3), and Beclin1, as well as their involvement in cadmium-induced autophagy and autophagic cell death. Renal injury, histological changes, and molecular marker of ER stress, FoxO3a, Bnip3, and autophagy were observed in the kidney cortex of CdCl2-exposed Sprague-Dawley rats. These observations indicate that ER stress, FoxO3a, Bnip3, and autophagy signaling were actively involved in cadmium-induced nephrotoxicity. Additionally, FoxO3a may act as a linking molecule to convey ER stress signals to Bnip3 and autophagy machinery upon cadmium exposure.

Herbal cake-partitioned moxibustion inhibits colonic autophagy in Crohn’s disease via signaling involving distinct classes of phosphatidylinositol 3-kinases

BACKGROUNDAutophagy is an evolutionarily conserved biological process in eukaryotic cells that involves lysosomal-mediated degradation and recycling of related cellular components. Recent studies have shown that autophagy plays an important role in the pathogenesis of Crohn’s disease (CD). Herbal cake-partitioned moxibustion (HM) has been historically practiced to treat CD. However, the mechanism by which HM regulates colonic autophagy in CD remains unclear.AIMTo observe whether HM can alleviate CD by regulating colonic autophagy and to elucidate the underlying mechanism.METHODSRats were randomly divided into a normal control (NC) group, a CD group, an HM group, an insulin + CD (I + CD) group, an insulin + HM (I + HM) group, a rapamycin + CD (RA + CD) group, and a rapamycin + HM (RA + HM) group. 2,4,6-trinitrobenzenesulfonic acid was administered to establish a CD model. The morphology of the colonic mucosa was observed by hematoxylin-eosin staining, and the formation of autophagosomes was observed by electron microscopy. The expression of autophagy marker microtubule-associated protein 1 light chain 3 beta (LC3B) was observed by immunofluorescence staining. Insulin and rapamycin were used to inhibit and activate colonic autophagy, respectively. The mRNA expression levels of phosphatidylinositol 3-kinase class I (PI3KC1), Akt1, LC3B, sequestosome 1 (p62), and mammalian target of rapamycin (mTOR) were evaluated by RT-qPCR. The protein expression levels of interleukin 18 (IL-18), tumor necrosis factor-α (TNF-α), nuclear factor κB/p65 (NF-κB p65), LC3B, p62, coiled-coil myosin-like BCL2-interacting protein (Beclin-1), p-mTOR, PI3KC1, class III phosphatidylinositol 3-kinase (PI3KC3/Vps34), and p-Akt were evaluated by Western blot analysis.RESULTSCompared with the NC group, the CD group showed severe damage to colon tissues and higher expression levels of IL-18 and NF-κB p65 in colon tissues (P < 0.01 for both). Compared with the CD group, the HM group showed significantly lower levels of these proteins (PIL-18 < 0.01 and Pp65 < 0.05). There were no significant differences in the expression of TNF-α protein in colon tissue among the rat groups. Typical autophagic vesicles were found in both the CD and HM groups. The expression of the autophagy proteins LC3B and Beclin-1 was upregulated (P < 0.01 for both) in the colon tissues of rats in the CD group compared with the NC group, while the protein expression of p62 and p-mTOR was downregulated (P < 0.01 for both). However, these expression trends were significantly reversed in the HM group compared with the CD group (PLC3B < 0.01, PBeclin-1 < 0.05, Pp62 < 0.05, and Pm-TOR < 0.05). Compared with those in the RA + CD group, the mRNA expression levels of PI3KC1, Akt1, mTOR, and p62 in the RA + HM group were significantly higher (PPI3KC1 < 0.01 and PAkt1, mTOR, and p62 < 0.05), while those of LC3B were significantly lower (P < 0.05). Compared with the RA + CD group, the RA + HM group exhibited significantly higher PI3KC1, p-Akt1, and p-mTOR protein levels (PPI3KC1 < 0.01, Pp-Akt1 < 0.05, and Pp-mTOR < 0.01), a higher p62 protein level (P = 0.057), and significantly lower LC3B and Vps34 protein levels (P < 0.01 for both) in colon tissue.CONCLUSIONHM can activate PI3KC1/Akt1/mTOR signaling while inhibiting the PI3KC3 (Vps34)-Beclin-1 protein complex in the colon tissues of CD rats, thereby inhibiting overactivated autophagy and thus exerting a therapeutic effect.

Protective Effects of Evogliptin on Steatohepatitis in High-Fat-Fed Mice

There are few studies on the effects of dipeptidyl peptidase-4 inhibitors on steatohepatitis. We explored whether evogliptin (Evo), a dipeptidyl peptidase-4 inhibitor, protects against steatohepatitis in a high-fat diet (HFD)-fed mice and whether these effects involve modulation of mitophagy. Adult male C57BL/J mice were divided into the normal diet (ND), HFD (45% of energy from fat) with Evo (250 mg/kg) (HFD + Evo), and HFD groups at 4 weeks of age and were sacrificed at 20 weeks of age. The HFD group showed hepatic lipid accumulation; this was decreased in the Evo + HFD group. There was an increased 8-hydroxydeoxyguanosine (8-OHDG) expression in the HFD group compared to ND mice. However, 8-OHDG expression levels were significantly decreased in the HFD + Evo group. Expressions of the mitophagy markers PTEN-induced kinase 1 (PINK1), Parkin, and BNIP-3 (BCL2 Interacting Protein 3) were significantly increased in the HFD group. However, the expressions of these markers were lower in the HFD + Evo group than that in the HFD group. Phospho-Akt was upregulated and p53 was downregulated in the HFD + Evo group compared to the HFD group. Evogliptin may alleviate steatohepatitis in HFD-fed mice by ameliorating steatosis and oxidative stress and by modulating mitophagy in the liver.

The Cardioprotective Role of Flaxseed in the Prevention of Doxorubicin- and Trastuzumab-Mediated Cardiotoxicity in C57BL/6 Mice

Although the combination of doxorubicin (DOX) and trastuzumab (TRZ) reduces the progression and recurrence of breast cancer, these anticancer drugs are associated with significant cardiotoxic side effects. We investigated whether prophylactic administration of flaxseed (FLX) and its bioactive components, α-linolenic acid (ALA) and secoisolariciresinol diglucoside (SDG), would be cardioprotective against DOX+TRZ-mediated cardiotoxicity in a chronic in vivo female murine model. Wild-type C57BL/6 female mice (10-12 wk old) received daily prophylactic treatment with one of the following diets: 1) regular control (RC) semi-purified diet; 2) 10% FLX diet; 3) 4.4% ALA diet; or 4) 0.44% SDG diet for a total of 6 wks. Within each arm, mice received 3 weekly injections of 0.9% saline or a combination of DOX [8mg/(kg.wk)] and TRZ [3mg/(kg.wk)] starting at the end of week 3. The main outcome was to evaluate the effects of FLX, ALA, and SDG on cardiovascular remodeling and markers of apoptosis, inflammation, and mitochondrial dysfunction. Significance between measurements was determined using a 4 (diet) × 2 (chemotherapy) × 2 (time) mixed factorial design with repeated measures. In the RC+DOX+TRZ-treated mice at week 6 of the study, the left ventricular ejection fraction (LVEF) decreased by 50% compared with the baseline LVEF (P<0.05). However, the prophylactic administration of the FLX, ALA, or SDG diet was partially cardioprotective, with mice in these treatment groups showing an ∼68% increase in LVEF compared with the RC+DOX+TRZ-treated group at week 6 (P<0.05). Although markers of inflammation (nuclear transcription factor κB), apoptosis [poly (ADP-ribose) polymerase-1 and the ratio of BCL2-associated X protein to B-cell lymphoma-extra large], and mitochondrial dysfunction (BCL2-interacting protein 3) were significantly elevated by approximately 2-fold following treatment with RC+DOX+TRZ compared with treatment with RC+saline at week 6, prophylactic administration of FLX, ALA, or SDG partially downregulated these signaling pathways. In a chronic in vivo female C57BL/6 mouse model of DOX+TRZ-mediated cardiotoxicity, FLX, ALA, and SDG were partially cardioprotective.

Beclin 2 negatively regulates innate immune signaling and tumor development.

Beclin 2 plays a critical role in metabolic regulation and obesity, but its functions in innate immune signaling and cancer development remain largely unknown. Here, we identified Beclin 2 as a critical negative regulator of inflammation and lymphoma development. Mice with homozygous ablation of BCL2-interacting protein 2 (Becn2) developed splenomegaly and lymphadenopathy and markedly increased ERK1/2 and NF-κB signaling for proinflammatory cytokine production. Beclin 2 targeted the key signaling kinases MEKK3 and TAK1 for degradation through an ATG9A-dependent, but ATG16L/Beclin 1/LC3-independent, autophagic pathway. Mechanistically, Beclin 2 recruited MEKK3 or TAK1 through ATG9A to form a complex (Beclin 2-ATG9A-MEKK3) on ATG9A+ vesicles upon ULK1 activation. Beclin 2 further interacted with STX5 and STX6 to promote the fusion of MEKK3- or TAK1-associated ATG9A+ vesicles to phagophores for subsequent degradation. Importantly, Becn2-deficient mice had a markedly increased incidence of lymphoma development, with persistent STAT3 activation. Myeloid-specific ablation of MEKK3 (Map3k3) completely rescued the phenotypes (splenomegaly, higher amounts of proinflammatory cytokines, and cancer incidence) of Becn2-deficient mice. Hence, our findings have identified an important role of Beclin 2 in the negative regulation of innate immune signaling and tumor development through an ATG9A-dependent, but ATG16L/Beclin 1/LC3-independent, autophagic pathway, thus providing a potential target for the treatment of inflammatory diseases and cancer.

Rosmarinic acid-induced apoptosis and cell cycle arrest in triple-negative breast cancer cells

Rosmarinic acid (RA) is a polyphenolic compound with various pharmacological properties, including, anti-inflammatory, immunomodulatory, and neuroprotective, as well as having antioxidant and anticancer activities. This study evaluated the effects and mechanisms of RA in two racially different triple-negative breast cancer (TNBC) cell lines. Results obtained show that RA significantly caused cytotoxic and antiproliferative effects in both cell lines in a dose- and time-dependent manner. Remarkably, RA induced cell cycle arrest-related apoptosis and altered the expression of many apoptosis-involved genes differently. In MDA-MB-231 cells, RA arrested the cells in the G0/G1 phase. In contrast, the data suggest that RA causes S-phase arrest in MDA-MB-468 cells, leading to a 2-fold increase in the apoptotic effect compared to MDA-MB-231 cells. Further, in MDA-MB-231 cells, RA significantly upregulated the mRNA expression of three genes: harakiri (HRK), tumor necrosis factor receptor superfamily 25 (TNFRSF25), and BCL-2 interacting protein 3 (BNIP3). In contrast, in the MDA-MB-468 cell line, the compound induced a significant transcription activation in three genes, including TNF, growth arrest and DNA damage-inducible 45 alpha (GADD45A), and BNIP3. Furthermore, RA repressed the expression of TNF receptor superfamily 11B (TNFRSF11B) in MDA-MB-231 cells in comparison to the ligand TNF superfamily member 10 (TNFSF10) and baculoviral IAP repeat-containing 5 (BIRC5) in MDA-MB-468 cells. In conclusion, the data suggest that the polyphenol RA may have a potential role in TNBC therapies, particularly in MDA-MB-468 cells.

Melatonin Attenuates Anoxia/Reoxygenation Injury by Inhibiting Excessive Mitophagy Through the MT2/SIRT3/FoxO3a Signaling Pathway in H9c2 Cells.

PurposeAutophagy caused by ischemia/reperfusion (I/R) increases the extent of cardiomyocyte damage. Melatonin (Mel) diminishes cardiac injury through regulating autophagy and mitochondrial dynamics. However, illustrating the specific role of mitophagy in the cardioprotective effects of melatonin remains a challenge. The aim of our research was to investigate the impact and underlying mechanisms of melatonin in connection with mitophagy during anoxia/reoxygenation (A/R) injury in H9c2 cells.MethodsH9c2 cells were pretreated with melatonin with or without the melatonin membrane receptor 2 (MT2) antagonist 4-P-PDOT, the MT2 agonist IIK7 and the sirtuin 3 (SIRT3) inhibitor 3-TYP for 4 hours and then subjected to A/R injury. Cell viability, cellular apoptosis, necrosis levels and oxidative markers were assessed. The expression of SIRT3 and forkhead box O3a (FoxO3a), mitochondrial function and the levels of mitophagy-related proteins were also evaluated.ResultsA/R injury provoked enhanced mitophagy in H9c2 myocytes. In addition, increased mitophagy was correlated with decreased cellular viability, increased oxidative stress and mitochondrial dysfunction in H9c2 cells. However, melatonin pretreatment notably increased cell survival and decreased cell apoptosis and oxidative response after A/R injury, accompanied by restored mitochondrial function. The inhibition of excessive mitophagy is involved in the cardioprotective effects of melatonin, as shown by the decreased expression of the mitophagy-related molecules Parkin, Beclin1, and BCL2-interacting protein 3-like (BNIP3L, best known as NIX) and decreased light chain 3 II/light chain 3 I (LC3 II/LC3 I) ratio and upregulation of p62 expression. Moreover, the decreased expression of SIRT3 and FoxO3a in A/R-injured H9c2 cells was abrogated by melatonin, but these beneficial effects were attenuated by the MT2 antagonist 4-P-PDOT or the SIRT3 inhibitor 3-TYP and enhanced by the MT2 agonist IIK7.ConclusionThese results indicate that melatonin protects H9c2 cells during A/R injury through suppressing excessive mitophagy by activating the MT2/SIRT3/FoxO3a pathway. Melatonin may be a useful candidate for alleviating myocardial ischemia/reperfusion (MI/R) injury in the future, and the MT2 receptor might become a therapeutic target.

Rosmarinic Acid‐Induced Apoptosis and Cell Cycle Arrest in Triple‐Negative Breast Cancer Cells

Rosmarinic acid (RA) is one of the potent polyphenolic compounds in rosemary (Rosmarinus officinalis) extract. The compound has shown various bioactive properties, including, anti‐inflammatory, immunomodulatory, anti‐mutagenic, hepatoprotective, and neuroprotective, as well as having antioxidant and anti‐cancer activity. This study evaluated the effects and mechanisms of RA in two triple‐negative breast cancer (TNBC) cell lines; MDA‐MB‐231(MM‐231) and MDA‐MB‐468 (MM‐468); which were derived from a Caucasian and an African American woman, respectively. In this study, RA significantly and similarly inhibited cell viability and proliferation in both cell lines in a dose‐ and time‐dependent manner. The results obtained show that RA differentially affected the two cell lines in cell cycle arrest‐related apoptosis and in the alteration of the expression of many apoptosis‐involved genes. In MM‐231 cells, RA arrested the cells in the G0/G1 phase. In contrast, the data suggest that RA causes S‐phase arrest in MM‐468 cells and leads to a 2‐fold greater apoptotic effect than in MM‐231 cells. In MM‐231 cells, RA significantly upregulated the mRNA expression of three genes; harakiri (HRK), tumor necrosis factor receptor superfamily 25 (TNFRSF25), and BCL2 interacting protein 3 (BNIP3). In contrast, in MM‐468 cell line, the compound induced a significant transcription activation in five genes, including (TNF), baculoviral IAP repeat‐containing 3 (BIRC3), receptor‐interacting serine/threonine‐protein kinase 2 (RIPK2), BNIP3, and growth arrest and DNA damage‐inducible 45 alpha (GADD45A). Furthermore, RA repressed the expression of TNF receptor superfamily 11B (TNFRSF11B) in MM‐231 cells in comparison to the ligand TNF superfamily member 10 (TNFSF10) in MM‐468 cells. In conclusion, the data suggest that the polyphenol RA may have therapeutic potential for TNBC. However, RA should be further evaluated for in‐vivo studies and in non‐cancer cells.Support or Funding InformationU54 RCMI

Stabilization of Hypoxia-Inducible Factors and BNIP3 Promoter Methylation Contribute to Acquired Sorafenib Resistance in Human Hepatocarcinoma Cells.

Despite sorafenib effectiveness against advanced hepatocarcinoma (HCC), long-term exposure to antiangiogenic drugs leads to hypoxic microenvironment, a key contributor to chemoresistance acquisition. We aimed to study the role of hypoxia in the development of sorafenib resistance in a human HCC in vitro model employing the HCC line HepG2 and two variants with acquired sorafenib resistance, HepG2S1 and HepG2S3, and CoCl2 as hypoximimetic. Resistant cells exhibited a faster proliferative rate and hypoxia adaptive mechanisms, linked to the increased protein levels and nuclear translocation of hypoxia-inducible factors (HIFs). HIF-1α and HIF-2α overexpression was detected even under normoxia through a deregulation of its degradation mechanisms. Proapoptotic markers expression and subG1 population decreased significantly in HepG2S1 and HepG2S3, suggesting evasion of sorafenib-mediated cell death. HIF-1α and HIF-2α knockdown diminished resistant cells viability, relating HIFs overexpression with its prosurvival ability. Additionally, epigenetic silencing of Bcl-2 interacting protein 3 (BNIP3) was observed in sorafenib resistant cells under hypoxia. Demethylation of BNIP3 promoter, but not histone acetylation, restored BNIP3 expression, driving resistant cells’ death. Altogether, our results highlight the involvement of HIFs overexpression and BNIP3 methylation-dependent knockdown in the development of sorafenib resistance in HCC. Targeting both prosurvival mechanisms could overcome chemoresistance and improve future therapeutic approaches.

Galectin-3 and TRIM16 coregulate osteogenic differentiation of human bone marrow-derived mesenchymal stem cells at least partly via enhancing autophagy.

The osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is critical for bone homeostasis. Here, we investigated the regulation of Galectin-3 and tripartite motif protein 16 (TRIM16) on osteogenic differentiation of hBMSCs through autophagy. Quantitative PCR (qPCR) and western blot were performed to determine the expression of osteogenic markers, autophagic markers, Galectin-3 and TRIM16. Short-hairpin RNAs (shRNAs) and overexpression plasmids were used to manipulate the expression of Galectin-3, TRIM16 and Unc-51 like autophagy activating kinase 1 (ULK1). Alkaline phosphatase (ALP) activity was measured by ALP staining assay. Calcium deposition in differentiated hBMSCs was assessed by Alizarin Red S staining. LC3 puncta formation was monitored by immunofluorescence staining. The interaction between indicated proteins was confirmed by co-immunoprecipitation (Co-IP) assay. Either Galectin-3 or TRIM16 knockdown led to impaired ALP activity, reduced calcium deposition, down-regulation of pro-osteogenic markers as well as restrained autophagy in osteogenic-induced hBMSCs. However, overexpression of Galectin-3 or TRIM16 promoted osteogenic differentiation of hBMSCs, which was then compromised by autophagy inhibition. Co-IP experiment demonstrated that TRIM16 associated with Galectin-3 through ULK1. Meanwhile, osteogenic induction enhanced the association between TRIM16 and ULK1 or coiled-coil myosin-like BCL2-interacting protein (Beclin1), and TRIM16 increased the stability of ULK1 and Beclin1. Moreover, either TRIM16 or ULK1 knockdown dampened the pro-osteogenic effect of Galectin-3, which elucidated that Galectin-3 mediated osteogenic differentiation was at least partly dependent on TRIM16 and ULK1. In summary, the present study revealed Galectin-3 and TRIM16 co-regulated osteogenic differentiation of hBMSCs at least partly via enhancing autophagy, which might provide a promising approach for osteoporosis treatment in future.

Activation of BNIP3-mediated mitophagy protects against renal ischemia\u2013reperfusion injury

Acute kidney injury (AKI) is a syndrome of abrupt loss of renal functions. The underlying pathological mechanisms of AKI remain largely unknown. BCL2-interacting protein 3 (BNIP3) has dual functions of regulating cell death and mitophagy, but its pathophysiological role in AKI remains unclear. Here, we demonstrated an increase of BNIP3 expression in cultured renal proximal tubular epithelial cells following oxygen-glucose deprivation-reperfusion (OGD-R) and in renal tubules after renal ischemia–reperfusion (IR)-induced injury in mice. Functionally, silencing Bnip3 by specific short hairpin RNAs in cultured renal tubular cells reduced OGD-R-induced mitophagy, and potentiated OGD-R-induced cell death. In vivo, Bnip3 knockout worsened renal IR injury, as manifested by more severe renal dysfunction and tissue injury. We further showed that Bnip3 knockout reduced mitophagy, which resulted in the accumulation of damaged mitochondria, increased production of reactive oxygen species, and enhanced cell death and inflammatory response in kidneys following renal IR. Taken together, these findings suggest that BNIP3-mediated mitophagy has a critical role in mitochondrial quality control and tubular cell survival during AKI.

ENOS deletion impairs mitochondrial quality control and exacerbates Western diet-induced NASH.

Dysregulated mitochondrial quality control leads to mitochondrial functional impairments that are central to the development and progression of hepatic steatosis to nonalcoholic steatohepatitis (NASH). Here, we identify hepatocellular localized endothelial nitric oxide synthase (eNOS) as a novel master regulator of mitochondrial quality control. Mice lacking eNOS were more susceptible to Western diet-induced hepatic inflammation and fibrosis in conjunction with decreased markers of mitochondrial biogenesis and turnover. The hepatocyte-specific influence was verified via magnetic activated cell sorting purified primary hepatocytes and in vitro siRNA-induced knockdown of eNOS. Hepatic mitochondria from eNOS knockout mice revealed decreased markers of mitochondrial biogenesis (PPARγ coactivator-1α, mitochondrial transcription factor A) and autophagy/mitophagy [BCL-2-interacting protein-3 (BNIP3), 1A/1B light chain 3B (LC3)], suggesting decreased mitochondrial turnover rate. eNOS knockout in primary hepatocytes exhibited reduced fatty acid oxidation capacity and were unable to mount a normal BNIP3 response to a mitophagic challenge compared with wild-type mice. Finally, we demonstrate that eNOS is required in primary hepatocytes to induce activation of the stress-responsive transcription factor nuclear factor erythroid 2-related factor 2 (NRF2). Thus, our data demonstrate that eNOS is an important regulator of hepatic mitochondrial content and function and NASH susceptibility.

Tetramethylpyrazine protects retinal ganglion cells against H2O2‑induced damage via themicroRNA‑182/mitochondrial pathway.

Glaucoma is the leading cause of irreversible blindness worldwide; the apoptosis of the retinal ganglion cells (RGCs) is a hallmark of glaucoma. Tetramethylpyrazine (TMP) is the main active component of Ligusticum wallichii Franchat, and has been demonstrated to improve a variety of injuries through its antioxidative and antiapoptotic properties. However, these effects of TMP on glaucoma have not been studied. The present study aimed to investigate the potential role of TMP in glaucoma and to elucidate its possible mechanisms responsible for these effects. An in vitro model was generated, in which primary RGCs (PRGCs) were treated with H2O2. Our study revealed that TMP protected against H2O2-induced injury to PRGCs, as evidenced by enhanced cell viability, reduced caspase 3 activity and decreased cell apoptosis. We also reported that TMP treatment inhibited reactive oxygen species (ROS) production and malondialdehyde levels, but upregulated the antioxidative enzyme superoxide dismutase. In particular, TMP significantly increased the expression of microRNA-182-5p (miR-182) in H2O2-treated PRGCs, which was selected as the target miRNA for further research. In addition, our findings suggested that the protective effects of TMP on H2O2-induced injury were attenuated by knockdown of miR-182. The results of a luciferase reporter assay demonstrated that Bcl-2 interacting protein 3 (BNIP3), an effector of mitochondria-mediated apoptosis, was a direct target of miR-182. In addition, TMP treatment significantly decreased the expression of BNIP3, Bax, cleaved-caspase-3 and cleaved-poly(ADP-ribose)polymerase, but increased that of Bcl-2. Also, TMP treatment decreased the release of cytochrome c from mitochondria and improved mitochondrial membrane potential in H2O2-treated RGCs. Of note, the inhibitory effects of TMP on the mitochondrial apoptotic pathway were suggested to be reversed by knockdown of miR-182. Collectively, our findings provide novel evidence that TMP protects PRGCs against H2O2-induced damage through suppressing apoptosis and oxidative stress via the miR-182/mitochondrial apoptotic pathway.

Impairment of HIF-1α-mediated metabolic adaption by NRF2-silencing in breast cancer cells

Hypoxia, a common element in the tumor environment, leads to Hypoxia-Inducible Factor-1α (HIF-1α) stabilization to modulate cellular metabolism as an adaptive response. In a previous study, we showed that inhibition of the nuclear factor erythroid 2-like-2 (NFE2L2; NRF2), a master regulator of many genes coping with electrophilic and oxidative stress, elevated the level of miR-181c and induced mitochondrial dysfunction in colon cancer cells. In this study, we demonstrate that NRF2-silencing hindered HIF-1α accumulation in hypoxic breast cancer cells and subsequently suppressed hypoxia-inducible expression of glycolysis-associated glucose transporter-1, hexokinase-2, pyruvate dehydrogenase kinase-1, and lactate dehydrogenase A. HIF-1α dysregulation in NRF2-silenced cancer cells was associated with miR-181c elevation. Overexpression of miR-181c in breast cancer cells blocked HIF-1α accumulation and diminished hypoxia-inducible levels of glycolysis enzymes, whereas the inhibition of miR-181c in NRF2-silenced cells restored HIF-1α accumulation. In a subsequent metabolomic analysis, hypoxic incubation increased the levels of metabolites involved in glycolysis and activated the pentose phosphate pathway (PPP) in control cells. However, these elevations were less pronounced in NRF2-silenced cells. In particular, hypoxic incubation increased the levels of amino acids, which implies a shift to catabolic metabolism, and the increased levels were higher in control cells than in NRF2-silenced cells. Concurrently, hypoxia activated BCL2 interacting protein 3 (BNIP3)-mediated autophagy in the control cells and miR-181c was found to be involved in this autophagy activation. Taken together, these results show that hypoxia-induced metabolic changes to glycolysis, the PPP, and autophagy are inhibited by NRF2-silencing through miR-181c-mediated HIF-1α dysregulation. Therefore, targeting NRF2/miR-181c could be an effective strategy to counteract HIF-1α-orchestrated metabolic adaptation of hypoxic cancer cells.

Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways.

BackgroundHypoxia induces cell apoptosis in the uterosacral ligaments of patients with pelvic organ prolapse by upregulation of hypoxia-inducible factor-1α (HIF-1α). This study aimed to investigate the effects of HIF-1α on human uterosacral ligament fibroblasts (hUSLFs) following treatment with the chemical inducer of hypoxia, cobalt chloride (CoCl2), and to explore the underlying mechanisms.Material/MethodsTen women who underwent hysterectomy for benign disease provided uterosacral ligament tissue for cell extraction. Following CoCl2 treatment, cell viability of isolated and cultured hUSLFs was evaluated by the MTT assay. JC-1 fluorescence mitochondrial imaging was used to study the change in mitochondrial membrane potential. Cell apoptosis and expression of apoptosis-associated proteins and collagen type I alpha 1 (COL1A1) were measured by flow cytometry, TUNEL and Western blot, respectively.ResultsHypoxia increased the expression of HIF-1α and increased cell apoptosis, decreased cell viability and expression levels of COL1A1. The JC-1 assay showed that the mitochondrial membrane potential was reduced and caspase-8, and -9 inhibitors partly reduced hUSLF apoptosis. HIF-1α treatment downregulated the expression of cellular FLICE inhibitory protein (c-FLIP), decoy receptor 2 (DcR2), and the ratio of Bcl-2 to Bax, and upregulated the expression tumor necrosis factor related apoptosis-inducing ligand (TRAIL), death receptor 5 (DR5) or TRAIL-R2, Fas, Bcl-2 interacting protein 3 (BNIP3), and cytochrome C, and increased the activation of caspase-3, caspase-8, and caspase-9, all of which were reversed by knockdown of HIF-1α.ConclusionsHIF-1α significantly induced apoptosis of hUSLFs through both the cell death receptor and the mitochondrial-associated apoptosis pathways.

The increased activity of a transcription factor inhibits autophagy in diabetic embryopathy

Maternal diabetes induces neural tube defects and stimulates the activity of the forkhead box O3 (Fox)O3a in the embryonic neuroepithelium. We previously demonstrated that deleting the FOXO3a gene ameliorates maternal diabetes-induced neural tube defects. Macroautophagy (hereafter referred to as "autophagy") is essential for neurulation. Rescuing autophagy suppressed by maternal diabetes in the developing neuroepithelium inhibits neural tube defect formation in diabetic pregnancy. This evidence suggests a possible link between FoxO3a and impaired autophagy in diabetic embryopathy. We aimed to determine whether maternal diabetes suppresses autophagy through FoxO3a, and if the transcriptional activity of FoxO3a is required for the induction of diabetic embryopathy. We used a well-established type 1 diabetic embryopathy mouse model, in which diabetes was induced by streptozotocin, for our invivo studies. To determine if FoxO3a mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium, we induced diabetic embryopathy in FOXO3a gene knockout mice and FoxO3a dominant negative transgenic mice. Embryos were harvested at embryonic day 8.5 to determine FoxO3a and autophagy activity and at embryonic day 10.5 for the presence of neural tube defects. We also examined the expression of autophagy-related genes. C17.2 neural stem cells were used for invitro examination of the potential effects of FoxO3a on autophagy. Deletion of the FOXO3a gene restored the autophagy markers, lipidation of microtubule-associated protein 1A/1B-light chain 3I to light chain 3II, in neurulation stage embryos. Maternal diabetes decreased light chain 3I-positive puncta number in the neuroepithelium, which was restored by deleting FoxO3a. Maternal diabetes also decreased the expression of positive regulators of autophagy (Unc-51 like autophagy activating kinase 1, Coiled-coil myosin-like BCL2-interacting protein, and autophagy-related gene 5) and the negative regulator of autophagy, p62. FOXO3a gene deletion abrogated the dysregulation of autophagy genes. Invitro data showed that the constitutively active form of FoxO3a mimicked high glucose in repressing autophagy. In cells cultured under high-glucose conditions, overexpression of the dominant negative FoxO3a mutant blocked autophagy impairment. Dominant negative FoxO3a overexpression in the developing neuroepithelium restored autophagy and significantly reduced maternal diabetes-induced apoptosis and neural tube defects. Our study revealed that diabetes-induced FoxO3a activation inhibited autophagy in the embryonic neuroepithelium. We also observed that FoxO3a transcriptional activity mediated the teratogenic effect of maternal diabetes because dominant negative FoxO3a prevents maternal diabetes-induced autophagy impairment and neural tube defect formation. Our findings suggest that autophagy activators could be therapeutically effective in treating maternal diabetes-induced neural tube defects.

Maternal Physical Activity and Sex Impact Markers of Hepatic Mitochondrial Health.

Maternal exercise and physical activity during the gestational period can be protective against maternal high-fat diet-induced hepatic steatosis in older offspring. However, it is unknown whether these protective effects are seen in younger offspring. In this study, we investigated whether maternal physical activity would attenuate maternal western diet (WD)-induced steatosis in young adult rats. Female Wistar rats (7-8 wk of age) were randomized into WD (42% fat, 27% sucrose) or normal chow diet (ND), and further randomized into physical activity (RUN) or sedentary (SED) conditions for a total of four groups. Dams returned to ND/SED conditions after parturition. Postweaning, offspring were maintained in ND/SED conditions for 18 wk. Maternal WD-induced increases in male offspring body mass was attenuated in the WD/RUN offspring (P < 0.05). Maternal WD feeding significantly increased hepatic steatosis in male (but not female offspring), which was not attenuated by maternal RUN. However, maternal RUN increased (P < 0.05) hepatic markers of mitochondrial biogenesis and mitophagy (mitochondrial transcription factor A, peroxisome proliferator activator receptor γ, and nuclear factor E2-related factor 2) in all offspring and the mitophagy marker BCL2-interacting protein 3 in WD/RUN offspring. Interestingly, hepatic markers of de novo lipogenesis (fatty acid synthase and acetyl coenzyme A carboxylase), mitophagy (autophagy-related gene 12:5, BCL2-interacting protein 3, P62, and LC3 II/I), and mitochondria biogenesis/content (mitochondrial transcription factor A and OXPHOS-Complex II) were significantly increased in female versus male offspring. Although maternal physical activity did not attenuate maternal WD-induced hepatic steatosis as has been previously reported in older adult offspring, it did significantly increase hepatic markers of mitochondrial biogenesis and mitophagy. Furthermore, female offspring had elevated hepatic markers of mitochondrial health, possibly explaining why female rats are protected against maternal WD-induced hepatic steatosis. Future studies are warranted to shed light on the time line of hepatic steatosis development under the influence of maternal physical activity.

Investigation of key autophagy-and mitophagy-related proteins and gene expression in BALF cells from patients with IPF and RA-ILD.

Idiopathic pulmonary fibrosis (IPF) is a chronic and irreversible interstitial lung disease with a poor prognosis and limited therapeutic options. Over the past decade, research efforts have focused on the pathogenetic mechanisms involved in this enigmatic lung disease. Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease often complicated by the development of interstitial lung disease (ILD), leading to high mortality and morbidity. Autophagy is a process regulating the turnover of subcellular components and organelles, and represents a major cellular homeostatic mechanism. Recent evidence suggests a role of autophagy and mitochondrial dysfunction in the development of IPF, focusing on lung fibroblasts and epithelial cells. The aim of this study was to examine the mRNA levels of molecules involved inthe autophagy pathway in bronchoalveolar lavage fluid (BALF)-derived cellsfrom patients with IPF in comparison topatients with RA demonstrating lung involvement (ILD) by RT-qPCR. The significant upregulation of BECLIN1 was observed in patients with RA-ILD compared with those with IPF. Other genes involved in the autophagy pathway were also examined, such as Unc-51 like autophagy activating kinase 1 (ULK1), BCL2 interacting protein 3 (BNIP3) and p62. No differences in the mRNA expression levels of these genes were observed. As regards the selective degradation of mitochondria and mitophagy, similar PTEN-induced putative kinase 1 (PINK1) and PARKIN; E3 ubiquitin ligase (PRKN) expression, as well as PINK1 protein levels, were observed. On the whole, the findings of this study demonstrate an increased expression of BECLIN1 in BALF cells from patients with RA-ILD compared with those from patients with IPF, while similar levels in other key molecules implicating in the autophagy pathway were observed in patients with IPF and RA-ILD.

AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells

ABSTRACTIn most cases, macroautophagy/autophagy serves to alleviate cellular stress and acts in a pro-survival manner. However, the effects of autophagy are highly contextual, and autophagic cell death (ACD) is emerging as an alternative paradigm of (stress- and drug-induced) cell demise. AT 101 ([-]-gossypol), a natural compound from cotton seeds, induces ACD in glioma cells as confirmed here by CRISPR/Cas9 knockout of ATG5 that partially, but significantly rescued cell survival following AT 101 treatment. Global proteomic analysis of AT 101-treated U87MG and U343 glioma cells revealed a robust decrease in mitochondrial protein clusters, whereas HMOX1 (heme oxygenase 1) was strongly upregulated. AT 101 rapidly triggered mitochondrial membrane depolarization, engulfment of mitochondria within autophagosomes and a significant reduction of mitochondrial mass and proteins that did not depend on the presence of BAX and BAK1. Conversely, AT 101-induced reduction of mitochondrial mass could be reversed by inhibiting autophagy with wortmannin, bafilomycin A1 and chloroquine. Silencing of HMOX1 and the mitophagy receptors BNIP3 (BCL2 interacting protein 3) and BNIP3L (BCL2 interacting protein 3 like) significantly attenuated AT 101-dependent mitophagy and cell death. Collectively, these data suggest that early mitochondrial dysfunction and HMOX1 overactivation synergize to trigger lethal mitophagy, which contributes to the cell killing effects of AT 101 in glioma cells.Abbreviations: ACD, autophagic cell death; ACN, acetonitrile; AT 101, (-)-gossypol; BAF, bafilomycin A1; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BH3, BCL2 homology region 3; BNIP3, BCL2 interacting protein 3; BNIP3L, BCL2 interacting protein 3 like; BP, Biological Process; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CC, Cellular Component; Con, control; CQ, chloroquine; CRISPR, clustered regularly interspaced short palindromic repeats; DMEM, Dulbecco’s Modified Eagle Medium; DTT, 1,4-dithiothreitol; EM, electron microscopy; ER, endoplasmatic reticulum; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GO, Gene Ontology; HAcO, acetic acid; HMOX1, heme oxygenase 1; DKO, double knockout; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LPL, lipoprotein lipase, MEFs, mouse embryonic fibroblasts; mPTP, mitochondrial permeability transition pore; MTG, MitoTracker Green FM; mt-mKeima, mito-mKeima; MT-ND1, mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; PI, propidium iodide; PRKN, parkin RBR E3 ubiquitin protein ligase; SDS, sodium dodecyl sulfate; SQSTM1/p62, sequestome 1; STS, staurosporine; sgRNA, single guide RNA; SILAC, stable isotope labeling with amino acids in cell culture; TFA, trifluoroacetic acid, TMRM, tetramethylrhodamine methyl ester perchlorate; WM, wortmannin; WT, wild-type

Sestrins are differentially expressed with age in the skeletal muscle of men: A cross-sectional analysis.

A gradual loss of skeletal muscle mass is a common feature of aging, leading to impaired insulin sensitivity and mobility. Sestrin1, 2, 3 are multifunctional proteins that regulate the mammalian target of rapamycin complex (mTORC1), autophagy and redox homeostasis. It is unclear how aging affects Sestrins and their downstream targets in human, therefore this study examined the basal expression of Sestrins in three age groups, young, middle-aged and older men and explored the mTORC1 pathway, autophagy markers and antioxidant regulation. Older men had less Sestrin1 and 3 protein and a different pattern of Sestrin2 electrophoretic mobility. The mRNA expression of SESN1 was upregulated in older men, but the discrepancy was not by microRNA expression. Although protein expressions of Sestrins were downregulated with aging, phosphorylation of AMP-dependent protein kinase (AMPKαThr172) and read-outs of mTORC1 activation, ribosomal protein S6 kinase 1 (p70S6K1Thr421/Ser424) and 4E-binding protein 1 (4E-BP1) mobility shift were unaltered. However, total p70S6K1 and 4E-BP1 were reduced in middle-aged and older men. The mRNA expressions of autophagic markers including microtubule-associated protein 1 light chain 3 (LC3) and BCL2 interacting protein 3 (BNIP3) were upregulated in middle-aged and older men. Although nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was upregulated in older men, the protein and mRNA expressions of its downstream antioxidants were either increased, decreased or unaltered. No clear relationship was observed between Sestrins and their downstream targets, yet it can be concluded that Sestrins proteins are clearly downregulated with aging.